Tetramethylenedisulfotetramine (TETS), commonly called tetramine or TETS, is a highly toxic convulsant with a parenteral LD50 of 0.1-0.3 mg/kg in mice or rats (Haskell and Voss, 1957; Voss et al., 1961; Casida et al., 1976). In adult humans, 7-10 mg is estimated as a lethal dose (Guan et al., 1993). TETS was used as a rodenticide until banned worldwide in the early 1990's (Whitlow et al., 2005; Banks et al., 2012). It is, however, still available illegally, and is responsible for accidental and intentional poisonings, predominantly in China (Croddy, 2004; Wu and Sun, 2004; Zhang et al., 2011), but also in other countries, including the United States (Barrueto et al., 2003). Between 1991 and 2010 over 14,000 cases of TETS intoxication were reported in China with 932 deaths (Li et al., 2011). Extreme toxicity, history of intentional mass poisonings, and the absence of a specific antidote raise concern that TETS is a potential chemical threat agent that could cause mass casualties if released accidentally or intentionally (Whitlow et al., 2005; Jett and Yeung, 2010).
Mild to moderate poisoning with TETS leads to headache and dizziness whereas severe intoxication produces status epilepticus and coma (Whitlow et al., 2005; Li et al., 2011). Animal studies demonstrate that TETS is active as a convulsant when administered orally, parenterally and intraventricularly. Sublethal seizures are not associated with evidence of cellular injury or neurodegeneration although there is delayed transient reactive astrocytosis and microglial activation (Zolkowska et al., 2012).
The primary convulsant mechanism of TETS has been thought to relate to blockade of GABAA receptors and the seizures induced in animals resemble those produced by other GABAA receptor antagonists including picrotoxin and pentylenetetrazol. Limited cellular physiological studies and results from [35S]t-butylbicyclophosphorothionate binding to brain membranes indicate that TETS inhibits GABAA receptors with an IC50 in the range of 1 μM (Squires et al., 1983; Esser et al., 1991; Ratra et al., 2001) and it is therefore comparable in potency to picrotoxin as an inhibitor of GABAA receptors (Squires et al., 1983; Cole and Casida, 1986; Ratra et al., 2001).
Cultured hippocampal neurons display synchronous spontaneous Ca2+ oscillations (Tanaka et al., 1996) that are driven by action potential-dependent synaptic transmission. Disruption of Ca2+ oscillations by environmental toxicants has been reported (Soria-Mercado et al., 2009; Cao et al., 2010; Choi et al., 2010; Pereira et al., 2010; Cao et al., 2011). Hippocampal neurons also exhibit spontaneous electrical discharges as they form functional neuronal networks. These discharges, as detected in extracellular recordings, consist of infrequent synchronized field potentials, mixed with more frequent desynchronized random action potentials (Cao et al., 2012; Frega et al., 2012). Synchronous Ca2+ oscillations and neuronal electrical firing co-occur (Jimbo et al., 1993) and are important in mediating neuronal development and activity dependent dendritic growth (Wayman et al., 2008). Genetic or environmental factors that interfere with neuronal transmission influence the overall neuronal networks activity (Kenet et al., 2007; Meyer et al., 2008; Shafer et al., 2008; Frega et al., 2012; Wayman et al., 2012). For example picrotoxin, a GABAA receptor antagonist, produces striking changes in network electric activity (Cao et al., 2012; Frega et al., 2012). Diisopropylfluorophosphate, an irreversible inhibitor of cholinesterase has also been shown to elicit status epileptics in rats. Hippocampal neurons dissociated from the brains of diisopropylfluorophosphate exposed rats display significantly higher intracellular Ca2+ concentration which appears to be dependent on the N-methy-D-aspartate receptors (Deshpande et al., 2010).
In the present study, using rapid throughput assays we characterized the influence of TETS on the Ca2+ dynamics and neuronal firing activity. Inasmuch as TETS induces changes in Ca2+ dynamics that are similar to those produced by the GABAA receptor antagonists picrotoxin and bicuculline, our results support the view that TETS acts as a GABAA receptor antagonist. Using rapid throughput Ca2+ measurement, we identified several agents that reduce or prevent the alterations in Ca2+ dynamics induced by TETS, suggesting several treatment strategies for TETS-induced seizures, including the GABAA receptor positive modulators diazepam and allopregnanolone. In preliminary studies with mice, we confirmed that these two agents do inhibit TETS-induced clonic seizures and progression to tonic seizures and death supporting that measurement of Ca2+ dynamics is likely useful for identifying novel targeted interventions for TETS poisoning.